The Candidate's overall career goal is to establish and maintain an independent proteomic and molecular biology-based program focused on investigation of events that mediate diabetic nephropathy (DN). DN is the leading cause of end-stage renal disease (ESRD). There are no current treatments that completely prevent progression to ESRD. This emphasizes the health-related importance for discovery of novel regulatory events as potential therapeutic targets for the diagnosis, prevention and treatment of DN. Tubulointerstitial fibrosis (TF) is a prominent feature of progressive DN that is mediated by transforming growth factor-beta (TGF-beta). Notch4 is a fascinating membrane-bound transcription regulator that was recently implicated in TGF signaling and renal disease. Our preliminary findings indicate that Notch4 activation inhibits TGF-beta signaling in cultured renal tubular cells and show decreased expression of Notch4 in tubular cells from diabetic mice as compared with controls. This suggests a hypothesis that down-regulation of Notch4 activity promotes the TGF-beta-mediated effects in DN. This project will develop Notch4-deficient diabetic mice. Renal pathophysiology experiments will be performed to delineate the in vivo role of Notch4 in diabetes-mediated TF and TGF signaling. It is anticipated that these animals will also provide a better model for the study of TF, as none of the current diabetic animal models exactly mimic the TF observed in diabetic humans. Accumulating evidence suggests a key role of TGF-mediated epithelial to mesenchymal transition (EMT) in diabetes-associated TF. We have recently developed models for TGF-mediated EMT in human kidney (HK) tubule cell lines. We will also develop HK models for glucose and advanced glycation end product (AGE)-mediated EMT. We will use these cellular models in combination with active Notch4 transfection and RNAi-mediated Notch4 knockdown to dissect the role of Notch4 in different components of signaling and transcription activity in EMT. Lastly, we will use state-of-the-art proteomic techniques with the Notch4-deficient diabetic mice to define Notch4-mediated protein expression and organelle localization in DN. In addition to molecular insight provided by these studies, the animal model and pathophysiology training obtained during this Career Development Award will provide valuable resources for the Candidate to expand his diabetes-related program and obtain independent-level funding.